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Haematococcus pluvialis chloroplast expression system and application thereof

A technology for the expression of Haematococcus pluvialis and chloroplasts, applied in the field of genetic engineering, can solve problems that hinder research and application, and achieve the effects of superior large-scale culture traits, enhanced antibacterial properties, and increased yield and yield

Active Publication Date: 2020-01-10
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research report on the chloroplast expression system of this algae, which seriously hinders the further research and application of this algae

Method used

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  • Haematococcus pluvialis chloroplast expression system and application thereof
  • Haematococcus pluvialis chloroplast expression system and application thereof
  • Haematococcus pluvialis chloroplast expression system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Cloning of endogenous fragments of Haematococcus pluvialis chlorophyll

[0035] According to the chloroplast genome of Haematococcus pluvialis measured in our laboratory, the following primers were designed and synthesized:

[0036] SEQ1-for: 5'-CGCGCAAGCGTTGAGGAAGGTG-3'

[0037] SEQ1-rev: 5'-CAACCCTTAAAAACTAAGTATAGAC-3'

[0038] SEQ2-for: 5'-GGGGCCCCTAGACCGAAGGCG-3'

[0039] SEQ2-rev: 5'-CCTCGCTGATTCACACGGGATTC-3'

[0040] SEQ3-for: 5'-AAAGCGCAACCTTTAAAGTAGCTG-3'

[0041] SEQ3-rev: 5'-ATGTTTGTTTATTTTTTATTTTTAAATCT-3'

[0042] SEQ4-for: 5'-AGCCCCAAGTATGGGCTGGGGC-3'

[0043]SEQ4-rev: 5'-TTATTGATTGTAACAAAGAATAGTGTC-3'

[0044] SEQ5-for: 5'-TAGTATAGAATAGAAGTCTAACAGT-3'

[0045] SEQ5-rev: 5'-GGGGGTGCCCCAGAACCACCTG-3'

[0046] SEQ6-for: 5'-TTTTATTTTTTAAAATCATATAAACGTTA-3'

[0047] SEQ6-rev: 5'-CGGAGCTCGGGGGCCCCCAG-3'

[0048] Wherein the amplification product of primer SEQ1-for and SEQ1-rev is SEQ ID NO:1; The amplification product of primer SEQ2-for and ...

Embodiment 2

[0055] Embodiment 2: Construction of empty vector for Haematococcus pluvialis chloroplast transformation

[0056] Design and synthesize the following primers:

[0057] bar-for: 5’-ATGAGCCCAGAACGACGCC-3’

[0058] bar-rev: 5'-TCATCAAATCTCGGTGACGGG-3'

[0059] Using the commercial carrier pSVB as a template, PCR amplification was carried out with primers bar-for and bar-rev. The reaction program was: 94°C for 5 minutes for pre-denaturation; 94°C for 1 min, 54°C for 30 sec, 72°C for 40 sec, a total of 35 cycles ; 72 ° C 5min extension. The PCR amplification product is about 555 bp, which is the herbicide resistance gene bar . After the fragments were subjected to agarose gel electrophoresis, the gel was recovered (Tiangen kit) and purified for later use.

[0060] Based on the above products, pMD18T was used as the starting vector to construct the homologous recombination vector of Haematococcus pluvialis chloroplast by homologous recombination method. Among them, pMD-SEQ2, p...

Embodiment 3

[0078] Example 3 Application of the carrier obtained according to the above examples in the transformation of Haematococcus pluvialis chloroplast

[0079] 1. Construction of Chloroplast Expression Vector of Haematococcus Pluvialls

[0080] Design and synthesize the following primers:

[0081] F1-for: 5’- CCTCTAGA ATG CATCATCACCATCACCAT GGTTTCGGTTGCAACGGTCCCTGG-3'

[0082] F1-rev: 5’- GGGATCC GAAATGGTGATGGTGATGGTGCAT-3'

[0083] Among them, F1-for carries Xba I restriction site and 6×His tag, F1-rev contains Bam HI restriction site. Using artificially synthesized antimicrobial peptide gene 1 as a template, PCR amplification was carried out with primers F1-for and F1-rev. The reaction program was: 94°C for 5 min for pre-denaturation; 94°C for 1 min, 55°C for 30 sec, 72°C for 20 sec , a total of 35 cycles; 72 ° C 5min extension. The PCR amplification product is about 220 bp, that is, the 5' end contains Xba Antimicrobial peptide sequence of I enzyme cleavage site...

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Abstract

The invention relates to the technical field of biological genetic engineering, in particular to a haematococcus pluvialis chloroplast expression system and application thereof. A sequence as shown inSEQ ID NO:1 and a sequence as shown in SEQ ID NO:2 on a haematococcus pluvialis chloroplast genome are used as homologous arms, a sequence as shown in SEQ ID NO:3 and sequence as shown in SEQ ID NO:4are used as promoters, a sequence as shown in SEQ ID NO:5 and a sequence as shown in SEQ ID NO:6 are used as a terminator, a bar gene is used as a selective marker gene to build a homologous recombination carrier, a particle bombardment is used for guiding the carrier to haematococcus pluvialis cells, and transgenic algal plants are obtained through screening of herbicide glyphosate. By adoptingthe stable haematococcus pluvialis chloroplast expression system, stable expression of exogenous genes in a chloroplast can be realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Haematococcus pluvialis chloroplast expression system and its application. Background technique [0002] Haematococcus pluvialis (Haematococcus pluvialis) is a single-celled green algae that lives in freshwater, belonging to Chlorophyta, Chlorophyceae, Volvox, Haematococcus, and Haematococcus. Although Haematococcus pluvialis is a single-celled organism, its life cycle is complex. Usually its growth process is divided into two stages, namely swimming cells and immobile cells. The motile cells are oval or oval in shape, ranging in size from about 5 μm to 20-30 μm. There is a large space between the protoplast and the cell wall, and the two are connected by many branched or unbranched cytoplasmic filaments, and the space between them is filled with transparent glue-like substances. Protoplasts are oval in shape, often with papillary protrusions at the front end. M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/821C12N15/8213C12N15/8214
Inventor 崔玉琳王寅初任庆敏王康任家利焦绪栋秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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